Few patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) achieve prolonged disease-free survival. Blinatumomab, a bispecific T-cell engaging antibody construct, transiently links CD3-positive T cells to CD19-positive B cells. This phase 2 study evaluated stepwise (9-28-112 μg/d with weekly dose increases; n = 23) or flat (112 μg/d; n = 2) dosing of blinatumomab by continuous infusion, with dexamethasone prophylaxis, in patients with relapsed/refractory DLBCL. Patients received a median of 3 prior lines of therapy. Median time since last regimen was 1.5 months. Seventeen patients ended treatment in cycle 1 (induction), 7 in cycle 2 (consolidation), and 1 in retreatment. Among 21 evaluable patients, the overall response rate after 1 blinatumomab cycle was 43%, including complete responses (CRs) in 19%. Three patients had late CR in follow-up without other treatment. The most common adverse events with stepwise dosing were tremor (48%), pyrexia (44%), fatigue (26%), and edema (26%). Grade 3 neurologic events with stepwise dosing were encephalopathy and aphasia (each 9%) and tremor, speech disorder, dizziness, somnolence, and disorientation (each 4%). Of 5 (22%) patients who discontinued stepwise dosing because of adverse events, 4 (17%) had neurologic events. Most neurologic events resolved. The flat-dose cohort was stopped because of grade 3 neurologic events in both patients. Blinatumomab monotherapy appears effective in patients with relapsed/refractory DLBCL, a heavily pretreated patient population with a high unmet medical need. Further studies need to define the optimal approach to achieve the target dose without early dropout. The study was registered at www.clinicaltrials.gov as #NCT01741792.

We describe the generation and characterization of a fusion protein consisting of a humanized anti-fibroblast-activating protein (anti-FAP) Ab and human TNF replacing the IgG1 CH2/CH3 Fc domain. The construct was generated by recombinant DNA technology and preserved its IgG1-derived dimeric structure with the TNF molecule linked as a dimer. Expression in CHO cells was optimized in serum-free medium under GMP conditions to achieve production levels up to 15 mg/liter. Recognition of the FAP Ag by the construct was as good as that by the parental anti-FAP Ab. TNF signaling was induce able via both TNF receptor types. When acting in solution, the Ab-linked TNF dimer exhibited a 10- to 20-fold lower activity compared with recombinant trimeric TNF. However, after binding to FAP-expressing cells, immobilized anti-FAP-TNF dimer was equivalent to membrane-anchored TNF with regard to bioactivity. Amplification of TNF-related pathways by mimicking the membrane-integrated TNF signaling was detectable in various systems, such as apoptosis induction or tissue factor production. The difference in TNF receptor type 1 and 2 signaling by the anti-FAP-TNF construct correlated well with its Ag-bound or -soluble status. Translating the approach into a xenograft animal model (BALB/c nu/nu mice), we demonstrated low toxicity with measurable antitumor efficacy for the TNF fusion protein after i.v. application. Immunohistochemical analysis of tumor sections showed restricted TNF-mediated macrophage recruitment to the targeted tissue in a time- and dose-dependent manner. These data warrant transfer of the anti-FAP-TNF immunocytokine into clinical trials for the treatment of FAP-positive tumors.

Immunotherapeutic targeting of G250/Carbonic anhydrase IX (CA‐IX) represents a promising strategy for treatment of renal cell carcinoma (RCC). The well characterized human‐mouse chimeric G250 (cG250) antibody has been shown in human studies to specifically enrich in CA‐IX positive tumors and was chosen as a carrier for site specific delivery of TNF in form of our IgG‐TNF‐fusion protein (cG250‐TNF) to RCC xenografts. Genetically engineered TNF constructs were designed as CH2/CH3 truncated cG250‐TNF fusion proteins and eucariotic expression was optimized under serum‐free conditions. In‐vitro characterization of cG250‐TNF comprised biochemical analysis and bioactivity assays, alone and in combination with Interferon‐γ (IFNγ). Biodistribution data on radiolabeled [125J] cG250‐TNF and antitumor activity of cG250‐TNF, alone and in combination with IFNγ, were measured on RCC xenografts in BALB/c nu/nu mice. Combined administration of cG250‐TNF and IFNγ caused synergistic biological effects that represent key mechanisms displaying antitumor responses. Biodistribution studies demonstrated specific accumulation and retention of cG250‐TNF at CA‐IX‐positive RCC resulting in growth inhibition of RCC and improved progression free survival and overall survival. Antitumor activity induced by targeted TNF‐based constructs could be enhanced by coadministration of low doses of nontargeted IFNγ without significant increase in side effects. Administration of cG250‐TNF and IFNγ resulted in significant synergistic tumoricidal activity. Considering the poor outcome of renal cancer patients with advanced disease, cG250‐TNF‐based immunotherapeutic approaches warrant clinical evaluation. © 2009 UICC

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD50 of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.

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